1. Field of the Invention
The present invention relates to a Rho target protein derived from Homo sapiens and more specifically relates to a gene encoding the same and a diagnostic agent containing the same.
2. BACKGROUND ART
A group of small GTP-binding proteins (G-proteins) with molecular weights of 20,000-30,000 with no subunit structure is observed in organisms. To date, over fifty or more members have been found as the superfamily of the small G-proteins in a variety of organisms, from yeast to mammals. The small G-proteins are divided into four families of Ras, Rho, Rab and the others based on homologies of amino acid sequences. It has been revealed that the small G-proteins control a variety of cellular functions. For example, the Ras protein is considered to control cell proliferation and differentiation, and the Rho protein is considered to control cell morphological change, adhesion and motility.
The Rho protein, having GDP/GTP-binding activity and intrinsic GTPase activity, is believed to be involved in cytoskeletal responses to extracellular signals such as lysophosphatidic acid (LPA) and certain growth factors. When the inactive GDP-binding Rho is stimulated, it is transformed to the active GTP-binding Rho protein (hereinafter referred to as "the activated Rho protein") by GDP/GTP exchange proteins such as Smg GDS, Dbl or Ost. The activated Rho protein then acts on target proteins to form stress fibers and focal contacts, thus inducing the cell adhesion and motility (Experimental Medicine, Vol. 12, No. 8, 97-102 (1994); Takai, Y. et al., Trends Biochem. Sci., 20, 227-231 (1995)). On the other hand, the intrinsic GTPase activity of the Rho protein transforms the activated Rho protein to the GDP-binding Rho protein. This intrinsic GTPase activity is enhanced by what is called GTPase-activating proteins (GAP) (Lamarche, N. & Hall, A. et al., TIG, 10, 436-440 (1994)).
The Rho family proteins, including RhoA, RhoB, RhoC, Rac1, Rac2 and Cdc42, share more than 50% sequence identity with each other. The Rho family proteins are believed to be involved in the biological responses inducing the formation of stress fibers and focal contacts in response to extracellular signals such as lysophosphatidic acid (LPA) and growth factors (A. J. Ridley & A. Hall, Cell, 70, 389-399 (1992); A. J. Ridley & A. Hall, EMBO J., 1353, 2600-2610 (1994)). The subfamily Rho is also considered to be implicated in physiological functions associated with cytoskeletal rearrangements, such as cell morphological change (H. F. Parterson et al., J. Cell Biol., 111, 1001-1007 (1990)), cell adhesion (Morii, N. et al., J. Biol. Chem., 267, 20921-20926 (1992); T. Tominaga et al., J. Cell Biol., 120, 1529-1537 (1993); Nusrat, A. et al., Proc. Natl. Acad. Sci. USA, 92, 10629-10633 (1995); Landanna, C. et al., Science, 271, 981-983 (1996)), cell motility (K. Takaishi et al., oncogene, 9, 273-279 (1994)), and cytokinesis (K. Kishi et al., J. Cell Biol., 120, 1187-1195 (1993); I. Mabuchi et al., Zygote, 1, 325-331 (1993)). In addition, it has been suggested that the Rho is involved in the regulation of smooth muscle contraction (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); M. Noda et al., FEBS Lett., 367, 246-250 (1995); M. Gong et al., Proc. Natl. Acad. Sci. USA, 93, 1340-1345 (1996)), and the expression of phosphatidylinositol 3-kinase (PI3 kinase) (J. Zhang et al., J. Biol. Chem., 268, 22251-22254 (1993)), phosphatidylinositol 4-phosphate 5-kinase (PI4,5-kinase) (L. D. Chong et al., Cell, 79, 507-513 (1994)) and c-fos (C. S. Hill et al., Cell, 81, 1159-1170 (1995)).
Recently, it has also be found that Ras-dependent tumorigenesis is suppressed when the Rho protein of which the amino acid sequence has been partly substituted is introduced to cells, revealing that the Rho protein plays an important role in Ras-induced transformation, that is, tumorigenesis (G. C. Prendergast et al., Oncogene, 10, 2289-2296 (1995); Khosravi-Far, R. et al., Mol. Cell. Biol., 15, 6443-6453 (1995); R. Qiu et al., Proc. Natl. Acad. Sci. USA, 92, 11781-11785 (1995); Lebowitz, P. et al., Mol. Cell, Biol., 15, 6613-6622 (1995)).
It has also been demonstrated that mutation of GDP/GTP-exchange proteins which act on the Rho protein results in cell transformation (Collard, J., Int. J. Oncol., 8, 131-138 (1996); Hart, M. et al., J. Biol. Chem., 269, 62-65 (1994); Horii, Y. et al., EMBO J., 13, 4776-4786 (1994)).
In addition, the Rho protein has been elucidated to be involved in cancer cell invasion, that is, metastasis (Yoshioka, K. et al., FEBS Lett., 372, 25-28 (1995)). The cancer cell invasion is closely dependent on changes in cancer cell activity to form cell adhesion. In this context, the Rho protein is also known to be involved in the formation of cell adhesion (see above Morii, N. et al. (1992); Tominaga, T. et al. (1993); Nusrat, A. et al. (1995); Landanna C. et al. (1996)).
Furthermore, the involvement of phosphoinositide kinases in Rho signaling was reported. Rho (Chong, L. D. et al., Cell, 79, 507-513, 1994) and Rac (Hartwig, J. H. et al., Cell, 82, 643-653, 1995), another member of Rho family low-molecular-weight G protein, were demonstrated to stimulate the synthesis of phosphatidylinositol bisphosphate (PIP2) in different cell systems. Since the binding of PIP2 is believed to regulate functions of many actin-associated proteins (Janmey, P. A., Ann. Rev. Physiol., 56, 169-191, 1994), its synthesis in subcellular localization may promote focal actin rearrangement. One of the proteins regulated by PIP2 is profilin, which makes a complex with actin monomer and releases actin upon PIP2 binding. Profilin also promotes actin filament assembly in the presence of thymosin .beta.4 (Pantaloni, D. and Carlier, M-F, Cell, 75, 1007-1014, 1993). Focal accumulation of profilin is, therefore, supposed to be important in actin reorganization (Theriot, J. A. and Mitchison, T. J., Cell, 75, 835-838, 1993).
The actin cytoskeleton plays an important role in cell motility, morphology, phagocytosis and cytokinesis. It is spatially and dynamically rearranged, which provides forces for morphological changes and cell surface movement in most eukaryotic cells. The rearrangement of actin is caused rapidly by extracellular stimuli and a series of actin-binding proteins are believed to act synergistically in polymerization, crosslinking and anchoring of actin filaments. The low-molecular-weight G protein Rho has been shown to be required for a variety of actin-dependent cellular processes such as platelet aggregation (Morii, N. et al., J. Biol. Chem., 267, 20921-20926, 1992), lymphocyte adhesion (Tominaga, T. et al., J. Cell. Biol., 120, 1529-1537, 1993), acceleration of cell motility (Takaishi, K. et al., Oncogene, 11, 39-48, 1995), and contractile ring formation and cytokinesis (Kishi, K. et al., J. Cell. Biol., 120, 1187-1195, 1993 and Mabuchi, I. et al., Zygote, 1, 325-331, 1993). In cultured fibroblasts, microinjection of Rho protein rapidly induces formation of the actin stress fibers or focal adhesion. In contrast, inactivation of Rho by a botulinum C3 extracellular enzyme (ADP-ribosyltransferase) inhibits this process (Ridley, A. J. and Hall, A., Cell, 70, 389-399, 1992). The treatment with the C3 extracellular enzyme also inhibits lysophosphatidic acid (LPA)-, endothelin- or GTP.gamma.S-induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin (Kumagai, N. et al., J. Biol. Chem., 268, 24535-24538, 1993; Rankin, S. et al., FEBS Lett., 354, 315-319, 1994; Ridley, A. J. and Hall, A., Cell, 70, 389-399, 1992; and Seckl, M.J. et al., J. Biol. Chem., 270, 6984-6990, 1995). These results indicate that Rho protein regulates signal transduction pathways linking the extracellular stimuli to the rearrangement of actine cytoskeleton.
Rho protein is believed to have many target molecules and regulate a number of the signal transduction pathways. Recently, several proteins have been reported as possible target molecules in mammals. These proteins are protein kinase N (PKN) (Watanabe, G. et al., Science, 271, 645-648, 1996; Amano, M. et al., Science 271, 648-650, 1996), rhophilin (Watanabe, G. et al., Science, 271, 645-648, 1996), citron (Madaule, P. et al., FEBS Lett., 377, 243-248, 1995), pl6OROCK (Ishizaki, T. et al., EMBO J., 15, 1885-1896, 1996), ROK.alpha. (Leung, T. et al., J. Biol. Chem., 270, 29051-29054, 1995), Rho-associated kinase (Matsui, T. et al., EMBO J., 15, 1885-1893 (1996), rhotekin (Reid, T. et al., J. Biol. Chem., 271, 9816-9822, 1996), myosin light-chain phosphatase (Kimura, K. et al., Science, 273, 245-248, 1996), murine mDia (Narumiya, Shu et al., Proceedings of Joint Annual Conference of The Japanese Society of Biochemistry and The Japanese Society of Molecular Biology, pp31 and 319, 1996). All these proteins bind to GTP-binding RhoA protein, except that citron binds also to GTP-binding Rac1 protein.
Recently, the following target proteins of Rho protein in Saccharomyces cerevisiae have been reported: Pkc1P (Nonaka, H. et al., EMBO J., 14, 5931-5938, 1995; Kamada, Y. et al., J. Biol. Chem., 271, 9193-9196, 1996), 1,3-.beta.-glucan synthesizing enzyme (Drgonova, J. et al., Science, 272, 277-279, 1996; Qadota, H. et al., Science, 272, 279-281) and Bni1p (Kohno, H. et al., EMBO J., 15, 6060-6068, 1996).
On the other hand, Bni1p of Saccharomyces cerevisiae (Kohno, H. et al., 1996, loc. cit.), Drosophila diaphanous (Castrillion, D. H. and Wasserman, S. A., Development, 120, 3367-3377, 1994), Drosophila cappuccino (Emons et al., Genes and Dev., 9, 2482-2494, 1995), murine formin (Woychick et al., Nature 346, 850-853, 1990) and murine mDia (Narumiya et al., 1996, loc. cit.) are known as proteins containing a poly-proline region and an FH-2 region.
However, there has been no report on a human Rho target protein which binds to profilin to regulate rearrangement of Rho protein and actin cytoskeleton insofar far as the inventors of the present invention know.